Progression through the cell cycle is a highly ordered process, and yet little is known about the mechanisms that ensure the proper timing of the various cell cycle events. From studies conducted with mutant cell lines it became clear that later cell cycle events can occur even when earlier ones fail to take place. This suggested that under normal conditions there must be regulatory mechanisms that ensure the coordinated execution of the different cell cycle events. In the past year, we have focused our attention on the regulatory mechanisms that govern cell cycle progression though mitosis. We have previously shown that in budding yeast, the Pds1 protein acts to inhibit the premature initiation of anaphase, when sister chromatids separate and migrate to the future daughter cells. This year we were able to demonstrate that Pds1 is not only an inhibitor of anaphase, but that it also controls the exit of mitosis, when the daughter cells physically separate (published in Genes and Development, 13:1950-1959, 1999). In this study we propose that the participation of Pds1 in the regulatory mechanism that controls mitotic exit links the exit from mitosis to the prior execution of anaphase, thus ensuring that progression though mitosis will occur in an orderly fashion.We are now targeting our efforts towards the elucidation of the molecular mechanism underlying mitotic cell cycle regulation. Specifically, we have designed a genetic screen in budding yeast that will reveal components of this regulatory pathway. In this screen, we are looking for mutants that are insensitive to Pds1?s inhibitory effect on anaphase initiation or the exit from mitosis. We expect that these mutations will be in proteins that interact directly with Pds1, as well as in downstream effectors of this regulatory pathway. We have completed the first part of the screen, in which we have isolated 1,300 conditional mutants in a strain harboring an inducible form of Pds1 that is resistant to proteolytic degradation. We are currently screening through this collection to identify mutants that under induced conditions do not arrest in mitosis in response to the presence of Pds1. In a complementing approach, we are currently looking for proteins that interact with Pds1 both biochemically, and via the yeast two hybrid screen. These studies are expected to shed light on the regulation of cell cycle progression not only in yeast, but in higher eukaryotes as well. - Cell Cycle; DNA damage checkpoint; Mitosis; Pds1p; S. cerevisiae; protein degradation;